Aromatase inhibitors

ABSTRACT

This invention provides azole derivatives, their pharmaceutical formulations, and their use in methods of inhibiting aromatase and treating or preventing estrogen-dependent diseases in mammals.

BACKGROUND OF THE INVENTION

Estrogens are synthesized from androgenic steroids. In the biosyntheticpathway for estrogen formation, aromatization is an essential step. Itis generally believed that if the aromatase enzyme could be effectivelyinhibited, a useful treatment for estrogen dependent disorders could beobtained (see Cancer Research, Vol. 42, Suppl. 8: 3261s (1982)).

Several estrogen dependent diseases exist which could be treated witharomatase inhibitors. These include breast cancer, endometriosis,polycystic ovarian disease, benign breast disease, and endometrialcancer. A beneficial effect of antiestrogens in the treatment of breastcancer has been well established (see Br. J. Cancer, 25, 270 (1971)).Two of the known aromatase inhibitors, testolactone andaminoglutethimide, have shown a beneficial effect in treatment of breastcancer. See Cancer Research, supra.

Endometriosis is characterized by an abnormal proliferation of theendometrium of the uterus. Since the endometrium is dependent onestradiol for its growth, an inhibitor of estrogen production shouldstop the progression of the disease.

Benign breast disease, or often called fibrocystic breast disease,appears to be dependent on ovarian steroids. See Cancer, 49, 2534(1982). Aromatase inhibitors have not been tried in this disease, butantiestrogens seem to be of benefit. See Obstet. Gynecol., 54, 80(1979).

Polycystic ovarian disease is one of the most common causes ofinfertility in women. The disease appears to result from an abnormalityin steroid metabolism, and the major form of therapy in this disease isthe antiestrogen, clomiphene. See Clin. Endocrinol., 12, 177 (1980).

It is the purpose of this invention to provide novel azole derivatives,their pharmaceutical formulations, and a method of inhibiting the enzymearomatase in mammals employing the azole derivatives. The invention thusprovides for the treatment or prevention of breast cancer and otherestrogen-dependent diseases.

SUMMARY OF THE INVENTION

This invention provides triazole and tetrazole compounds of the formula##STR1##

wherein R is pyridyl or ##STR2##

R₁, R₂, R₃, and R₄ are independently hydrogen, methyl, trifluoromethyl,methoxy, or halo; and

E and Q are independently N or CH, with the proviso that E and Q may notboth be N at the same time, or a pharmaceutically acceptable saltthereof.

In addition, this invention provides a method of inhibiting aromatase inmammals which comprises administering an aromatase inhibiting amount ofa compound of the above formula. By virtue of their ability to inhibitthe enzyme aromatase, the compounds of the above formula are useful inthe treatment and prevention of estrogen-dependent diseases, espciallybreast cancer, in mammals.

Further provided by this invention are pharmaceutical formulationscomprising one or more of the compounds of the above formula incombination with a suitable pharmaceutical carrier, diluent, orexcipient therefor. The formulations provided by this invention areparticularly useful in treating mammals suffering fromestrogen-dependent diseases such as breast cancer.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENT

The term "halo" refers to fluoro, chloro, bromo, and iodo. "Pyridyl"refers to 2-, 4-, or especially 3-pyridyl.

A preferred group of compounds useful in this invention are thosewherein:

(a) R₁ is halo, especially fluoro or chloro, or trifluoromethyl, and

(b) R is phenyl (R₃ and R₄ are both hydrogen) or substituted phenyl,especially where R₃ is halo, especially chloro or fluoro, ortrifluoromethyl.

Especially preferred compounds are those wherein

(a) R₁ and R₂ are both chloro, especially 3,4-dichloro, and R isunsubstituted phenyl, or

(b) is mono-substituted phenyl, R₁ and R₃ are independently chloro orfluoro, and R₂ and R₄ are each hydrogen.

The most preferred compounds provided by this invention are1-[bis(4-chlorophenyl)methyl]-1H-1,2,3-triazole and2-[bis(4-chlorophenyl)methyl]-2H-tetrazole, and pharmaceuticallyacceptable salts thereof.

As will be recognized by those skilled in the art, the compounds of theabove formula may contain an asymmetric carbon atom. This invention isnot limited to any particular isomer but includes the individualenantiomers as well as the racemates of the compounds.

In addition, when one of E and Q is N and the other is CH, the compoundsof the above formula are 1- or 2-substituted tetrazoles. Although eachof these two types of tetrazoles are claimed individually, because ofthe method of preparation, the two isomers may be formed together. Thus,this invention includes the individual 1- and 2-substituted tetrazolesand mixtures thereof. This latter mixture is referred to herein as1(2)-substituted tetrazoles.

The pharmaceutically acceptable acid addition salts of the basesrepresented by the above formula can be prepared employing those acidsof sufficient acidity to form acid addition salts. These include bothinorganic and organic acids such as hydrochloric, hydrobromic,hydriodic, sulfuric, phosphoric, oxalic, methanesulfonic,benzenesulfonic, p-toluenesulfonic, maleic, and the like acids.Preferred acids for salt formation are the inorganic acids, especiallyhydrochloric acid.

The compounds of this invention may be prepared by any of severalmethods. The preferred method of preparing these compounds is summarizedby the following scheme: ##STR3## where X is halo, preferably chloro orbromo, or hydroxy. Generally, compounds II and III are allowed to reacttogether in the presence of a base, such as an alkali metal hydride,hydroxide, alkoxide, or carbonate, preferably in the presence of anon-reactive high boiling solvent, such as dioxane, a dialkylformamide,such as dimethylformamide, and the like. Typically, an excess of theazole derivative II is employed, usually between a slight excess and a6-fold molar excess. The reaction is generally carried out under aninert atmosphere and at temperatures from about 20° C. up to the boilingpoint of the reaction mixture. Under the preferred reaction conditionsof sodium hydride and dimethylformamide at about 80°-100° C., thereaction is usually complete within about 2-24 hours. Intermediates IIand III are either commercially available, are known in the literature,or can be prepared by methods known in the art.

In order to more fully illustrate the preperation of the compounds ofthis invention, the following examples are provided. The examples areillustrative only and are not intended to limit the scope of theinvention in any way.

EXAMPLE 1 2-[Bis(4-chlorophenyl)methyl]-2H-tetrazole A. Preparation of4,4'-dichlorobenzhydryl chloride

Forty-two milliliters of thionyl chloride were gradually added to 75 gof 4,4'-dichlorobenzhydrol. When the addition was complete, the reactionwas heated on a steam bath for 2 hours. The excess thionyl chloride wasremoved in vacuo. One hundred milliliters of carbon tetrachloride wereadded, and the solvent was again evaporated. The carbon tetrachloridetreatment was repeated and upon evaporation the resulting oil wascrystallized from peotroleum ether to provide 65.3 g of the desiredsub-titled intermediate, m.p. 63° C.

B. Preparation of 2-[bis(4-chlorophenyl)methyl]-2H-tetrazole

A mixture of 3.12 g of tetrazole, 26.2 g of potassium carbonate, and 10g of 4,4'-dichlorobenzhydryl chloride from above in 150 ml ofdimethylformamide was heated under a nitrogen atmosphere for 2.5 hoursat 85°-90° C. and then overnight at 60°-65° C. The reaction mixture waspoured into 1 liter of a saturated aqueous sodium chloride solutionwhich was then extracted twice with 200 ml portions of ethyl acetate.The combined organic extracts were washed three times with a saturatedsodium chloride solution, dried over magnesium sulfate, and evaporatedto dryness to provide 12.1 g of the crude product as a pale yellow oil.The residue was purified by high pressure liquid chromatography. Theprimary product which was recovered was 1.1 g of the 2-tetrazolederivative as white crystals when recrystallized from methanol, m.p.91°-92° C. Mass spectral analysis confirmed that the product had thecorrect molecular formula (M⁺ =305) and ¹³ C and ¹ H NMR NOE experimentsconfirmed that the primary product was2-[bis(4-chlorophenyl)methyl]-2H-tetrazole.

Analysis for C₁₄ H₁₀ Cl₂ N₄ : Calculated: C, 55.10; H, 3.30; N, 18.36;Found: C, 55.05; H, 3.45; N, 18.18.

EXAMPLE 2 1-[bis(4-chlorophenyl)methyl]-1H-1,2,3-triazole

Following the general procedure of Example 1B, 5.0 g of 1,2,3-triazolein 100 ml of dimethylformamide were treated first with 1.44 g of sodiumhydride (60% in mineral oil), and then with 5.0 g of4,4'-dichlorobenzhydryl chloride. The reactants were stirred overnightat about 25° C. and then heated for 1 hour on a steam bath.Chromatography of the residue after work-up provided 2.4 g of theundesired 2-[bis(4-chlorophenyl)methyl]-2H-1,2,3-triazole and 3.1 g ofthe desired title product, m.p. 124°-125° C.

Analysis for C₁₅ H₁₁ Cl₂ N₃ : Calculated: C, 59.23; H, 3.65; N, 13.81;Found: C, 59.45; H, 3.86; N, 13.81.

The compounds provided by this invention are useful in preventing ortherapeutically treating estrogen-dependent diseases, including breastcancer, in mammals by virtue of their ability to inhibit the enzymearomatase. Their ability to inhibit aromatase was demonstrated byemploying a modification of the isolated rat ovarian microsome method ofBrodie et al. in J. Steroid Biochem., 7, 787 (1976). In this testsystem, ovarian microsomes are obtained from rats treated with pregnantmares serum gonadotropin. Test compounds are added to reaction vialscontaining 0.1 μM 4-androstene-3,17-dione, 100,000 dpm 1,2[³H]-androstenedione, the microsomes and a NADPH generating system. Theconcentrations of the inhibitors tested ranged between 0.005 and 10 μM.In this assay, aromatization of androstenedione results in theproduction of [³ H]-H₂ O which is isolated by extracting the sampleswith chloroform and treating the aqueous phase with charcoal to removethe free steroid. Samples are counted in a liquid scintillationspectrometer and the percent inhibition determined by comparing theresults with control samples incubated without inhibitor. Potency isdetermined based on the concentration of inhibitor in μM required toproduce a 50% inhibition of enzyme activity (EC₅₀) when theconcentration of substrate (androstenedione) is 0.1 μM. The EC₅₀ 's ofcertain of the compounds of the above formula are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Aromatase Inhibition in the Rat                                               Ovarian Microsome Assay                                                       Compound           EC.sub.50 *                                                ______________________________________                                        1-[bis(4-chlorophenyl)-                                                                          <0.05                                                      methyl]-1H--1,2,3-triazole                                                    2-[bis(4-chlorophenyl)-                                                                           0.15                                                      methyl]-2H--tetrazole                                                         ______________________________________                                         *Concentration of compound in μM required to achieve 50% inhibition of     aromatase activity when substrate concentration is 0.1 μM.            

The compounds may be administered by any number of routes, including theoral, subcutaneous, intramuscular, intravenous, transdermal, and rectalroutes. The compounds are usually employed in the form of pharmaceuticalcompositions. Such compositions are prepared in a manner well known inthe pharmaceutical art and comprise at least one active compound of theabove formula.

Accordingly, the invention includes a pharmaceutical compositioncomprising as active ingredient a compound of the above formulaassociated with a pharmaceutically acceptable carrier. In making thecompositions of the present invention, the active ingredient willusually be mixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the composition can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,emulsions, solutions, syrups, suspensions, aerosols (as a solid or in aliquid medium), ointments containing for example up to 10% by weight ofthe active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, tragacanth, gelatin, syrup,methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesiumstearate, water, and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Thecompositions of the invention may be formulated so as to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart.

For oral administration, a compound of this invention can be admixedwith carriers and diluents molded into tablets or enclosed is gelatincapsules. The mixtures can alternatively be dissolved in liquids such asten percent aqueous glucose solution, isotonic saline, sterile water, orthe like, and administered intravenously or by injection. Such solutionscan, if desired, be lyophilized and stored in a sterile ampoule readyfor reconstitution by the addition of sterile water for readyintramuscular injection.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 1 to about 500 mg, more usually about 5 toabout 300 mg, of the active ingredient. The term "unit dosage form"refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalcarrier.

The active compounds are effective over a wide dosage range. Forexample, dosages per day will normally fall within the range of about0.05 to about 300 mg/kg of body weight. In the treatment of adulthumans, the range of about 0.1 to about 50 mg/kg, in single or divideddoses, is preferred. However, it will be understood that the amount ofthe compound actually administered will be determined by a physician, inthe light of the relevant circumstances including the condition to betreated, the choice of compound to be administered, the age, weight, andresponse of the individual patient, the severity of the patient'ssymptoms, and the chosen route of administration, and therefore theabove dosage ranges are not intended to limit the scope of the inventionin any way.

In order to more fully illustrate the operation of this invention, thefollowing formulation examples are provided. The examples areillustrative only and are not intended to limit the scope of theinvention. The formulations employ as active compounds any of thepharmaceutical compounds of the above formula.

EXAMPLE 3

Hard gelatin capsules are prepared using the following ingredients:

    ______________________________________                                                           per capsule                                                ______________________________________                                        1-[bis(4-fluorophenyl)methyl]-1H--                                                                 250 mg                                                   1,2,3-triazole                                                                Starch dried         200 mg                                                   Magnesium stearate    10 mg                                                   Total                460 mg                                                   ______________________________________                                    

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

EXAMPLE 4

Capsules each containing 20 mg of medicament are made as follows:

    ______________________________________                                                           per capsule                                                ______________________________________                                        1(2)-[(4-chlorophenyl)(4-fluoro-                                                                   20 mg                                                    phenyl)methyl]-1H(2H)--tetrazole                                              Starch               89 mg                                                    Microcrystalline cellulose                                                                         89 mg                                                    Magnesium stearate    2 mg                                                    Total                200 mg                                                   ______________________________________                                    

The active ingredient, cellulose, starch and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve and filled into hardgelatin capsules in 200 mg quantities.

EXAMPLE 5

Capsules each containing 100 mg of active ingredient are made asfollows:

    ______________________________________                                                            per capsule                                               ______________________________________                                        1-[(4-trifluoromethylphenyl)(3-                                                                     100      mg                                             pyridyl)methyl]-1H--1,2,3-triazole                                            Polyoxyethylenesorbitan monooleate                                                                  50       mcg                                            Starch powder         250      mg                                             ______________________________________                                    

The above ingredients are thoroughly mixed and are placed in an emptygelatin capsule.

EXAMPLE 6

Tablets each containing 10 mg of active ingredient are made up asfollows:

    ______________________________________                                                           per tablet                                                 ______________________________________                                        1(2)-[(2,4-dichlorophenyl)(4-                                                                      10 mg                                                    chlorophenyl)methyl]-1H(2H)--                                                 tetrazole                                                                     Starch               45 mg                                                    Microcrystalline cellulose                                                                         35 mg                                                    Polyvinylpyrrolidone  4 mg                                                    (as 10% solution in water)                                                    Sodium carboxymethyl starch                                                                        4.5 mg                                                   Magnesium stearate   0.5 mg                                                   Talc                  1 mg                                                    Total                100 mg                                                   ______________________________________                                    

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate and talc, previouslypassed through a No. 60 mesh U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yield tabletseach weighing 100 mg.

EXAMPLE 7

A tablet formula is prepared using the ingredients below:

    ______________________________________                                                          per tablet                                                  ______________________________________                                        1-[1-(4-chlorophenyl)-1-phenyl-                                                                   250 mg                                                    ethyl]-1H--1,2,3-triazole                                                     Cellulose microcrystalline                                                                        400 mg                                                    Silicon dioxide fumed                                                                              10 mg                                                    Stearic acid         5 mg                                                     Total               665 mg                                                    ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg.

EXAMPLE 8

Suppositories each containing 25 mg of active ingredient are made asfollows:

    ______________________________________                                                          per suppository                                             ______________________________________                                        1-[(4-methylphenyl)(4-methoxy-                                                                      25 mg                                                   phenyl)methyl]-1H--1,2,3-triazole                                             Saturated fatty acid glycerides to                                                                2,000 mg                                                  ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

EXAMPLE 9

Suspensions each containing 5 mg of medicament per 5 ml dose are made asfollows:

    ______________________________________                                                          per 5 ml of suspension                                      ______________________________________                                        1(2)-[(3-pyridyl)(3,4-difluoro-                                                                   5          mg                                             phenyl)methyl]-1H(2H)--tetrazole                                              Sodium carboxymethyl cellulose                                                                    50         mg                                             Syrup               1.25       ml                                             Benzoic acid solution                                                                             0.10       ml                                             Flavor              q.v.                                                      Color               q.v.                                                      Purified water to   5          ml                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxyethylcellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor and color is diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

EXAMPLE 10

An aerosol solution is prepared containing the following components:

    ______________________________________                                                        Weight %                                                      ______________________________________                                        1-(diphenylmethyl)-1H--                                                                          0.25                                                       1,2,3-triazole                                                                Ethanol           29.75                                                       Propellant 22     70.00                                                       (Chlorodifluoromethane)                                                       ______________________________________                                    

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to -30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted further with the remaining amount of propellant.The valve units are then fitted to the container.

We claim:
 1. A method of inhibiting aromatase in a mammal whichcomprises administering to said mammal an aromatase inhibiting amount ofa compound of the formula ##STR4## wherein R is pyridyl or ##STR5## R₁,R₂, R₃, and R₄ are independently hydrogen, methyl, trifluoromethyl,methoxy, or halo; andE and Q are independently N or CH, with the provisothat E and Q may not both be N at the same time, or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1 employing a compoundwherein R is ##STR6##
 3. The method according to claim 2 employing acompound wherein R₁ and R₃ are independently chloro or fluoro.
 4. Themethod according to claim 3 employing1-[bis(4-chlorophenyl)methyl]-1H-1,2,3-triazole or a pharmaceuticallyacceptable salt thereof.
 5. The method according to claim 3 employing2-[bis(4-chlorophenyl)methyl]-2H-tetrazole or a pharmaceuticallyacceptable salt thereof.
 6. A method of preventing or treatingestrogen-dependent diseases in a mammal which comprises administering tosaid mammal an effective amount of a compound of the formula ##STR7##wherein R is pyridyl or ##STR8## R₁, R₂, R₃, and R₄ are independentlyhydrogen, methyl, trifluoromethyl, methoxy, or halo; andE and Q areindependently N or CH, with the proviso that E and Q may not both be Nat the same time, or a pharmaceutically acceptable salt thereof.
 7. Themethod according to claim 6 employing1-[bis(4-chlorophenyl)methyl]-1H-1,2,3-triazole or a pharmaceuticallyacceptable salt thereof.
 8. The method according to claim 6 employing2-[bis(4-chlorophenyl)methyl]-2H-tetrazole or a pharmaceuticallyacceptable salt thereof.
 9. The method according to claim 6 wherein theestrogen-dependent disease is breast cancer.
 10. The method according toclaim 9 employing 1-[bis(4-chlorophenyl)methyl]-1H-1,2,3-triazole or apharmaceutically acceptable salt thereof.
 11. The method according toclaim 9 employing 2-[bis(4-chlorophenyl)methyl]-2H-tetrazole or apharmaceutically acceptable salt thereof.
 12. A pharmaceuticalformulation which comprises a compound of the formula ##STR9## wherein Ris pyridyl or ##STR10## R₁, R₂, R₃, and R₄ are independently hydrogen,methyl, trifluoromethyl, methoxy, or halo; andE and Q are independentlyN or CH, with the proviso that E and Q may not both be N at the sametime, or a pharmaceutically acceptable salt thereof, in combination witha suitable pharmaceutical carrier, diluent, or excipient therefor.
 13. Aformulation according to claim 12 employing a compound wherein R is##STR11## R₁ and R₃ are independently chloro or fluoro, and R₂ and R₄are each hydrogen.
 14. A formulation according to claim 13 employing1-[bis(4-chlorophenyl)methyl]-1H-1,2,3-triazole or a pharmaceuticallyacceptable salt thereof.
 15. A formulation according to claim 13employing 2-[bis(4-chlorophenyl)methyl]-2H-tetrazole or apharmaceutically acceptable salt thereof.